Mosaic HIV envelope immunogenic polypeptides

ABSTRACT

Disclosed herein are mosaic HIV envelope (Env) polypeptides that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). Also disclosed are sets of the disclosed mosaic Env polypeptides, which include two or more (for example, three) of the polypeptides. Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in a subject comprising administering to the subject at least one (such as two, three, or more) of the immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the §371 U.S. National Stage of International Application No.PCT/US2014/058443, filed Sep. 30, 2014, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/884,696, filed Sep. 30, 2013, which isincorporated by reference herein in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Contract No.DE-AC52-06NA25396 awarded by the U.S. Department of Energy and grantnumber AI100645 from the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

This disclosure relates to immunogenic polypeptides, particularlypolypeptides that can elicit an immune response to humanimmunodeficiency virus (HIV) in a subject.

BACKGROUND

Approximately 35 million people worldwide are estimated to be infectedwith human immunodeficiency virus (HIV). Although infection rates aredeclining, in 2012 about 2.3 million people were newly infected andabout 1.6 million people died from AIDS-related illnesses. However,viral diversity in HIV and the occurrence of escape variants providesignificant challenges to development of effective HIV vaccines. Thus,there remains a need for the development of effective vaccines to treatand inhibit HIV infection worldwide.

SUMMARY

Disclosed herein are mosaic HIV envelope (Env) proteins that can elicitan immune response to HIV (such as cytotoxic T cell (CTL), helper Tcell, and/or humoral responses). In specific examples, the disclosedmosaic proteins (also referred to herein as mosaic Env proteins orimmunogenic polypeptides) can elicit B cell responses to HIV. Alsodisclosed are sets of mosaic Env proteins, which include two or more(for example, three or more) of the proteins. In some embodiments, thedisclosed mosaic Env proteins or sets of mosaic Env proteins areincluded in an immunogenic composition, such as a polyvalent immunogeniccomposition.

Also disclosed herein are methods for treating or inhibiting HIV in asubject including administering one or more of the disclosed immunogenicpolypeptides or compositions to a subject infected with HIV or at riskof HIV infection. In some embodiments, the methods include inducing animmune response to HIV in a subject, comprising administering to thesubject at least one (such as two, three, or more) of the disclosedimmunogenic polypeptides or at least one (such as two, three, or more)nucleic acids encoding at least one of the immunogenic polypeptidesdisclosed herein.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing an exemplary approach to mosaic Bcell epitope design.

FIGS. 2A-2D are a series of diagrams showing the draft Ca trace of theunliganded HIV -1_(JR-FL) Env trimer. The Ca trace is shown as abackbone worm. The backbone worms of all three protomers are shown inFIGS. 2A and 2B, and the backbone worm of one protomer is shown in FIGS.2C and 2D. In FIGS. 2A and 2C, the Env trimer is viewed from theperspective of the target cell. In FIGS. 2B and 2D, the trimer is shownfrom a perspective parallel to the viral membrane. The density map isshown as a blue mesh.

FIG. 3 is a plot showing 10 amino acid cluster coverage per strain byeach of the trivalent polypeptide options. For each trivalentpolypeptide option, the strains are shown in the order (left to right):C clade, CRF07 CRF08 (China C), CRF01, CRF02, A, B, D, F, G, Global. Thefirst two on the left of each group are C clade and China CRF07 andCFR08, that are basically C clade in Env, respectively. Within clade,three naturals do very well in terms of C clade potential epitopecoverage; however the C clade mosaic performs slightly better. Theinter-clade coverage is poor for either of these C clade-specificcompositions. An advantage of the M group design (which is based on theglobal diversity or HIV-1, rather than a single clade) is that the Cclade coverage is comparable to within-clade, but all other clades aresimultaneously well covered, so the potential to be a global vaccine isenhanced.

FIGS. 4A-4C are a series of plots showing titration of Mmos3.1 (FIG.4A), Mmos3.2 (FIG. 4B), and Mmos 3.3 (FIG. 4C) with the HIV-neutralizingmonoclonal antibody PG9, using surface plasmon resonance (SPR) (Hearty,Methods Mol. Biol. 907:411-42, 2012). The slow off rate of PG9 whenbound to Mos3.2 is indicated by the gradual decline after the peak.

FIG. 5 shows the relative binding of Mmos3.1 to 17b, a monoclonalantibody which is CD4-inducible and mimics the CCR5 co-receptor binding,after binding to CD4. The graph shows the ratio of 17b relative to ConSafter binding to A32, sCD4, and T8. ConS is an HIV consensus proteinthat is particularly sensitive to CD4 induction of the 17b binding site.A32 is a monoclonal antibody that mimics CD4 in this process, and T8 isa control.

FIG. 6 shows the relative binding of Mmos3.2 to 17b compared with ConSafter binding to A32, sCD4, and T8, as described in FIG. 5.

FIG. 7 shows the relative binding of Mmos3.3 to 17b compared with ConSafter binding to A32, sCD4, and T8 as described in FIG. 5.

FIG. 8 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2, andMmos 3.3 binding to CD4 binding site targeting neutralizing antibodyVRC01.

FIG. 9 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2, andMmos 3.3 with V3 region antibody 19b.

FIG. 10 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2,and Mmos 3.3 with HIV V1-V2 glycan binding neutralizing antibody CH02.CH01 failed to bind to the mosaic proteins.

FIG. 11 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2,and Mmos 3.3 with V2-region clade specific antibody 697D.

FIG. 12 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2,and Mmos 3.3 with HIV carbohydrate binding neutralizing antibody 2G12.

FIG. 13 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2,and Mmos 3.3 with the HIV specific monoclonal antibody CH58.

FIG. 14 shows a series of plots of SPR titration of Mmos3.1, Mmos3.2,and Mmos 3.3 with the potent HIV neutralizing antibody PGT128.

SEQUENCE LISTING

The nucleic acid and amino acid sequences disclosed herein and in theaccompanying Sequence Listing are shown using standard letterabbreviations for nucleotide bases, and one letter code for amino acids.Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Mar. 22, 2016,and is 59,531 bytes, which is incorporated by reference herein.

SEQ ID NOs: 1-8 are the amino acid sequence of exemplary Env mosaicproteins.

SEQ ID NO: 9 is the amino acid sequence of a tissue plasminogenactivator leader peptide.

DETAILED DESCRIPTION

I. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” Thus,“comprising an antigen” means “including an antigen” without excludingother elements.

It is further to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described below. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the disclosure, thefollowing explanations of terms are provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in mineral oil (Freund incomplete adjuvant), sometimeswith the inclusion of killed mycobacteria (Freund's complete adjuvant)to further enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Immunostimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants includebiological molecules (a “biological adjuvant”), such as costimulatorymolecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF,TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.Adjuvants can be used in combination with the disclosed immunogenicpolypeptides.

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intravenous, thecomposition (such as a disclosed antigen) is administered by introducingthe composition intravenously into a subject.

Antibody: A polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or fragments thereof, which specifically bindsand recognizes an analyte (such as an antigen or immunogen) such as aEnv polypeptide or antigenic fragment thereof. Immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable regiongenes.

Antibodies exist, for example as intact immunoglobulins and as a numberof well characterized fragments produced by digestion with variouspeptidases. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) thatbind to Env would be Env-specific binding agents. This includes intactimmunoglobulins and the variants and portions of them well known in theart, such as Fab′ fragments, F(ab)′₂ fragments, single chain Fv proteins(scFv), and disulfide stabilized Fv proteins (dsFv). A scFv protein is afusion protein in which a light chain variable region of animmunoglobulin and a heavy chain variable region of an immunoglobulinare bound by a linker, while in dsFvs, the chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.The term antibody also includes genetically engineered forms such aschimeric antibodies (such as humanized murine antibodies),heteroconjugate antibodies (such as bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York,1997.

Antibody fragments are defined as follows: (1) Fab, the fragment whichcontains a monovalent antigen-binding fragment of an antibody moleculeproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (3) (Fab′)₂, the fragment of the antibody obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (SCA), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. The term “antibody” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and lightchain contains a constant region and a variable region, (the regions arealso known as “domains”). In combination, the heavy and the light chainvariable regions specifically bind the antigen. Light and heavy chainvariable regions contain a “framework” region interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs.” The extent of the framework region and CDRs have been defined(see, Kabat et al., Sequences of Proteins of Immunological Interest,U.S. Department of Health and Human Services, 1991, which is herebyincorporated by reference). The Kabat database is now maintained online.The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. Light chain CDRs are sometimes referred to as CDRL1, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to asCDR H1, CDR H2, and CDR H3.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas.” Monoclonal antibodies include humanized monoclonalantibodies.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in a subject, includingcompositions that are injected or absorbed into a subject. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedantigens. “Epitope” or “antigenic determinant” refers to the region ofan antigen to which B and/or T cells respond. In one embodiment, T cellsrespond to the epitope, when the epitope is presented in conjunctionwith an MHC molecule. Epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5, about 9, about 8-10, or about 6-22 amino acids in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and nuclearmagnetic resonance.

Examples of antigens include, but are not limited to, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell. In some examples, antigensinclude peptides derived from a pathogen of interest. Exemplarypathogens include bacteria, fungi, viruses and parasites. In specificexamples, an antigen is derived from HIV, for example, one or more HIVpolypeptides or a fragment thereof, such as at least a portion of an Envprotein.

B cell: A lymphocyte, a type of white blood cell that expressesimmunoglobulin on its surface and can ultimately develop into anantibody secreting a plasma cell. In one example, a B cell expressesCD19 (CD19+). An “immature B cell” is a cell that can develop into amature B cell. Generally, pro-B cells (that express, for example, CD45or B220) undergo immunoglobulin heavy chain rearrangement to becomepro-B pre-B cells, and further undergo immunoglobulin light chainrearrangement to become an immature B cells. Immature B cells include T1and T2 B cells. Immature B cells express IgM on their cell surface andcan develop into mature B cells, which can express different forms ofimmunoglobulin (e.g., IgA, IgG). B cells can be activated by agents suchas lipopolysaccharide (LPS), CD40 ligation, and antibodies thatcrosslink the B cell receptor (immunoglobulin), including antigen, oranti-Ig antibodies. Neutralizing antibodies can inhibit HIV infection ofthe natural viral target cell, CD4 positive T cells.

Envelope (Env): The envelope protein from HIV. Env is initiallysynthesized as a precursor protein of 845-870 amino acids in size(gp160). gp160 forms a homotrimer and undergoes glycosylation in theGolgi apparatus. It is then cleaved by a cellular protease into gp120and gp41. gp120 includes most of the surface-exposed domains of the Envglycoprotein complex and binds to the cellular CD4 receptor and cellularchemokine receptors (for example, CCR5). Env is the only HIV proteincapable of stimulating HIV neutralizing antibodies.

HXB2 numbering system: A reference numbering system for HIV protein andnucleic acid sequences, which uses HIV-1 HXB2 strain sequences as areference for all other HIV strain sequences. The person of ordinaryskill in the art is familiar with the HXB2 numbering system (Korber etal., Human Retroviruses and AIDS 1998: A Compilation and Analysis ofNucleic Acid and Amino Acid Sequences. Korber B, Kuiken C L, Foley B,Hahn B, McCutchan F, Mellors J W, and Sodroski J, Eds.; TheoreticalBiology and Biophysics Group, Los Alamos National Laboratory, LosAlamos, N.M., incorporated by reference herein in its entirety). HXB2 isalso known as: HXBc2, for HXB clone 2; HXB2R, in the Los Alamos HIVdatabase, with the R for revised, as it was slightly revised relative tothe original HXB2 sequence; and HXB2CG in the NCBI GenBank database, forHXB2 complete genome.

Host cells: Cells in which a virus or vector can be propagated and itsDNA expressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immunogenic polypeptide: A protein or a portion thereof that is capableof inducing an immune response in a subject, such as a subject infectedwith, or at risk of infection with, a pathogen. Administration of animmunogenic polypeptide derived from a pathogen of interest can elicitan immune response against the pathogen. Administration of animmunogenic polypeptide can lead to protective immunity against apathogen of interest (such as HIV). In some examples, an immunogenicpolypeptide is a polypeptide including one or more regions from an HIVproteome, for example, an Env protein, such as a mosaic Env protein.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies. Some HIV neutralizing antibody responses can betype-specific, and only elicit responses that neutralize a singlestrain, while others are broad and can elicit responses to manydifferent strains.

Immunogenic composition: A composition comprising an immunogenicpolypeptide or a nucleic acid encoding an immunogenic polypeptide thatinduces a measurable CTL response against virus expressing theimmunogenic polypeptide or a portion thereof, induces a measurablehelper T cell response, or induces a measurable B cell response (such asproduction of antibodies) against the immunogenic polypeptide or aportion thereof. In one example, an “immunogenic composition” iscomposition including one or more polypeptides from HIV, such as one ormore of the mosaic Env proteins disclosed herein. It further refers toisolated nucleic acids encoding an immunogenic polypeptide, such as anucleic acid that can be used to express the immunogenic polypeptide(and thus be used to elicit an immune response against this polypeptideor a portion thereof).

For in vitro use, an immunogenic composition may consist of at least one(such as two or more) isolated polypeptides, peptide epitopes, ornucleic acids encoding the polypeptide or peptide epitope. For in vivouse, the immunogenic composition will typically include at least one(such as one, two, three, or more) polypeptide, peptide, or nucleic acidin pharmaceutically acceptable carriers, and/or other agents. Anyparticular peptide, such as a disclosed polypeptide or a nucleic acidencoding the polypeptide, can be readily tested for its ability toinduce a CTL, helper T cell, or B cell response by art-recognizedassays. Immunogenic compositions can include adjuvants, which are wellknown to one of skill in the art.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as acquired immune deficiency syndrome (AIDS), AIDS-relatedconditions, HW infection (such as HIV-1 infection), or combinationsthereof. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological condition(such as AIDS or AIDS related conditions) after it has begun to develop.The term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, an improvement in the overall healthor well-being of the subject, or by other parameters well known in theart that are specific to the particular disease. A “prophylactic”treatment is a treatment administered to a subject who does not exhibitsigns of a disease or exhibits only early signs for the purpose ofdecreasing the risk of developing pathology.

Isolated: An “isolated” biological component (such as a protein, forexample a disclosed polypeptide or nucleic acid encoding such apolypeptide) has been substantially separated or purified away fromother biological components in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA, RNA, and proteins.Proteins, peptides, and nucleic acids that have been “isolated” includeproteins purified by standard purification methods. The term alsoembraces proteins or peptides prepared by recombinant expression in ahost cell as well as chemically synthesized proteins, peptides, andnucleic acid molecules.

Isolated does not require absolute purity, and can include protein,peptide, or nucleic acid molecules that are at least 50% isolated, suchas at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Mosaic polypeptide or mosaic protein: A polypeptide or protein assembledfrom fragments of natural sequences via computational optimization(e.g., Fischer et al., Nat. Med. 13:100-106, 2007). Multiple sequences(for example, thousands of sequences) are used as input and thesequences are evolved by recombination in silico. Recombinants areconstrained to have natural breakpoints and a mosaic set is designed tomaximize coverage of potential epitopes (such as B cell epitopes) for aviral population.

Operably linked: A first nucleic acid is operably linked with a secondnucleic acid when the first nucleic acid is placed in a functionalrelationship with the second nucleic acid. For instance, a promoter isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked nucleic acids are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame. In some examples, theoperably linked nucleic acids are heterologous, for example, the firstand second nucleic acids are from different organisms, different genes,or different polypeptides and the resulting nucleic acid is notnaturally occurring.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington: TheScience and Practice of Pharmacy, The University of the Sciences inPhiladelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia,Pa., 21^(st) Edition (2005), describes compositions and formulationssuitable for pharmaceutical delivery of the proteins, nucleic acids, andother compositions herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions, powder, pill, tablet, or capsule forms,conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polypeptide: Any compound composed of amino acids and/or amino acidanalogs, chemically bound together. Polypeptide as used herein includesoligomers of amino acids and/or amino acid analogs, or small and largepeptides, including proteins. Any chain of amino acids, regardless oflength or post-translational modification (such as glycosylation orphosphorylation) is referred to as a polypeptide. The term polypeptideapplies to amino acid polymers including naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers as well aspolymers in which one or more amino acid residue is a non-natural aminoacid, for example an artificial chemical mimetic of a correspondingnaturally occurring amino acid. As used herein, polypeptide also refersto recombinant amino acid polymers, such as polymers including portionsthat are obtained from different (typically non-contiguous) portions ofa genome (such as an HIV genome) and/or are obtained from differentgenomes (such as two or more HIV strains). A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic.

Polyvalent immunogenic composition: A composition including two or moreseparate immunogenic polypeptides (such as a “cocktail” of immunogenicpolypeptides) that are capable of eliciting an immune response in asubject, for example an immune response to HIV. In some examples, apolyvalent immunogenic composition includes two or more immunogenicpolypeptides (or nucleic acids encoding the polypeptides). In onespecific example, a polyvalent immunogenic composition includes threeEnv proteins, such as three Env mosaic proteins disclosed herein.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified protein isone in which the protein is more enriched than the protein is in itsnatural environment within a cell. Preferably, a preparation is purifiedsuch that the protein represents at least 50% of the protein content ofthe preparation.

Recombinant nucleic acid or polypeptide: A nucleic acid molecule orpolypeptide that is not naturally occurring or has a sequence that ismade by an artificial combination of two otherwise separated segments ofnucleotide or amino acid sequence. This artificial combination isaccomplished by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acids, e.g., by genetic engineeringtechniques such as those described in Sambrook et al. (ed.), MolecularCloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The term “recombinant”includes nucleic acids or polypeptides that have been altered solely byaddition, substitution, or deletion of a portion of a natural nucleicacid molecule or peptide.

Sequence identity/similarity: Sequence identity between two or morenucleic acid sequences or between two or more amino acid sequences canbe measured in terms of percentage identity; the higher the percentage,the more identical the sequences are. Homologs or orthologs of nucleicacid or amino acid sequences possess a relatively high degree ofsequence identity/similarity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations. The NCBI BasicLocal Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403-10, 1990) is available from several sources, including theNational Center for Biological Information (NCBI, National Library ofMedicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on theInternet, for use in connection with the sequence analysis programsblastp, blastn, blastx, tblastn, and tblastx. Blastn is used to comparenucleic acid sequences, while blastp is used to compare amino acidsequences. Additional information can be found at the NCBI web site.

In some examples, sequence similarity is assessed by the conservation ofepitope-length fragments. The use of this measure of similarity wasdeveloped at Los Alamos National Laboratory, and tools are available onthe World Wide Web at hiv.lanl.gov.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals (including non-humanprimates).

Therapeutically effective amount or effective amount: The amount of anagent, such as a nucleic acid, polypeptide, or other therapeutic agent,that is sufficient to prevent, treat (including prophylaxis), reduceand/or ameliorate the symptoms and/or underlying causes of a disorder ordisease, for example to prevent, inhibit, and/or treat HIV. In someembodiments, an “effective amount” is sufficient to reduce or eliminatea symptom of a disease, such as AIDS. For instance, this can be theamount necessary to inhibit viral replication or to measurably alteroutward symptoms of the viral infection, such as an increase of T cellcounts in the case of an HIV infection. In general, this amount will besufficient to measurably inhibit virus (for example, HIV) replication orinfectivity. An “anti-viral agent” or “anti-viral drug” is an agent thatspecifically inhibits a virus from replicating or infecting cells.Similarly, an “anti-retroviral agent” is an agent that specificallyinhibits a retrovirus from replicating or infecting cells.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

Vaccine: A pharmaceutical composition that elicits a prophylactic ortherapeutic immune response in a subject. In some cases, the immuneresponse is a protective immune response and can block subsequentinfection, in other cases it can limit the pathological impact of aninfection by containing the infection. Typically, a vaccine elicits anantigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen, or to a cellular constituent correlated with apathological condition. A vaccine may include a polynucleotide (such asa nucleic acid encoding a disclosed antigen), a peptide or polypeptide(such as a disclosed antigen), a virus, a cell, or one or more cellularconstituents.

Vector: A nucleic acid molecule that can be introduced into a host cell,thereby producing a transformed host cell. Recombinant DNA vectors arevectors having recombinant DNA. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. Viral vectors arerecombinant DNA vectors having at least some nucleic acid sequencesderived from one or more viruses.

Virus: A virus consists essentially of a core of nucleic acid surroundedby a protein coat, and has the ability to replicate only inside a livingcell. “Viral replication” is the production of additional virus by theoccurrence of at least one viral life cycle. A virus may subvert thehost cell's normal functions, causing the cell to behave in a mannerdetermined by the virus. For example, a viral infection may result in acell producing a cytokine, or responding to a cytokine, when theuninfected cell does not normally do so. In some examples, a virus is apathogen.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), andfeline immunodeficiency virus (FIV).

HIV-1 is a retrovirus that causes immunosuppression in humans (HIVdisease), and leads to a disease complex known as the acquiredimmunodeficiency syndrome (AIDS). “HIV disease” refers to awell-recognized constellation of signs and symptoms (including thedevelopment of opportunistic infections) in persons who are infected byan HIV virus, as determined by antibody or western blot studies ordetection of HIV nucleic acids. Laboratory findings associated with thisdisease are a progressive decline in T cells.

II. Description of Several Embodiments

Disclosed herein are mosaic polypeptides from HIV Env protein (alsoreferred to herein as immunogenic polypeptides). The mosaic polypeptides(also referred to as mosaic proteins) are computationally designed tooptimally cover global HIV diversity (e.g., having the potential toelicit broadly cross-reactive immune responses), as described inExamples 1 and 2, below. Mosaic polypeptides are assembled fromfragments of natural sequences via a computational optimization method(e.g., U.S. Pat. App. Publ. No. 2012/0231028, incorporated herein byreference in its entirety). In some embodiments, mosaic polypeptidesresemble natural proteins, but do not exist in nature. Thousands ofsequences are use used as input, and the sequences are evolved byrecombination in silico. Recombinants are constrained to have naturalbreakpoints, and a mosaic set will maximize the coverage of potentialepitopes (such as B cell epitopes) for a viral population. Combinationsof mosaics are selected to give the optimal coverage of potentialepitopes found in natural sequences for a given number of mosaics. TheEnv mosaic proteins and nucleic acids encoding the proteins disclosedherein are capable of eliciting an immune response to HIV in a subject.

Also disclosed herein are sets of the immunogenic polypeptides (such assets of two or more polypeptides) that can be used to elicit an immuneresponse to HIV in a subject. B cells undergo a process called affinitymaturation, where they evolve to improve affinity for their targetepitope during the immune response. Without being bound by theory, it isbelieved that by administering a set of Env polypeptides includingcoverage of multiple HIV variants to a subject, exposing a B celllineage to common variants of an HIV epitope during affinity maturationmay yield an antibody response with greater breadth and ability tointeract with natural variants, by selecting for antibodies that highaffinity for the most common forms of the epitope.

A. Immunogenic Polypeptides

Exemplary amino acid sequences of HIV Env mosaic proteins identified asdescribed in Example 2 include SEQ ID NOs: 1-8 disclosed herein. In someexamples, the disclosed polypeptides include, consist essentially of, orconsist of an amino acid sequence at least 95% identical to the aminoacid sequence set forth as one of SEQ ID NOs: 1-8, such as at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical, or even 100% identical to thesequence set forth as one of SEQ ID NOs: 1-8.

In some embodiments, the disclosed Env mosaic proteins are utilized incombination, for example as sets of immunogenic polypeptides. In someexamples, the set of Env mosaic proteins includes 2, 3, 4, 5, 6, 7, or 8of the disclosed polypeptides. The sets of polypeptides are selected forproviding coverage of variants within a single HIV clade (for example,within clade C), providing coverage of variants between clades (forexample, at least clade B, clade C, and CRF01), and/or global coverage(for example M group), and can be administered to a subject, for exampleas a polyvalent immunogenic composition. Exemplary sets of polypeptidesare shown in Table 1. However, additional combinations of the disclosedimmunogenic polypeptides can also be selected to produce additionalsets.

TABLE 1 Exemplary sets of Env mosaic polypeptides Set PolypeptidesWithin Clade C SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 Three clade SEQID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 Global SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8

In some examples, a leader or signal peptide is linked to theimmunogenic polypeptide, for example to increase expression and/orimmunogenicity of the polypeptide. In one example, the leader peptide isa tissue plasminogen activator (tPA) leader peptide, for example, apeptide having the amino acid sequence MDAMKRGLCCVLLLCGAVFVSAR (SEQ IDNO: 9). One of skill in the art can identify other suitable leaderpeptides that can be used to optimize expression of the disclosedpolypeptides.

The immunogenic polypeptides disclosed herein can be chemicallysynthesized by standard methods, or can be produced recombinantly, forexample by expression of the polypeptide from a nucleic acid moleculethat encodes the polypeptide. An exemplary process for polypeptideproduction is described in Lu et al., FEBS Lett. 429:31-35, 1998. Theycan also be isolated by methods including preparative chromatography andimmunological separations.

B. Nucleic Acids

Nucleic acids encoding the disclosed Env mosaic proteins (e.g., SEQ IDNOs: 1-8) are also disclosed herein. Unless otherwise specified, a“nucleic acid encoding a polypeptide” includes all nucleotide sequencesthat are degenerate versions of each other and encode the same aminoacid sequence. For example, a polynucleotide encoding a disclosedimmunogenic polypeptide includes a nucleic acid sequence that isdegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Therefore,all degenerate nucleotide sequences are included as long as the aminoacid sequence of the polypeptide encoded by the nucleotide sequence isunchanged. In some embodiments, the disclosed polypeptide sequences areback-translated to codon optimized DNA using standard methods.

The nucleic acids encoding a disclosed polypeptide include a recombinantDNA which is incorporated into a vector, such as an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (such as a cDNA)independent of other sequences. Methods for the manipulation andinsertion of the nucleic acids of this disclosure into vectors are wellknown in the art (see for example, Sambrook et al., Molecular Cloning, aLaboratory Manual, 2d edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989, and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N.Y., 1994). DNA sequences encoding the polypeptide can be expressed invitro or in vivo by DNA transfer into a suitable host cell. The cell maybe prokaryotic or eukaryotic. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

Polynucleotide sequences encoding the disclosed polypeptides can beoperatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is joined suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

Hosts can include microbial, yeast, insect and mammalian organisms.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archaea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, Neurospora, and immortalized mammalian myeloid andlymphoid cell lines. Techniques for the propagation of mammalian cellsin culture are well-known (see, Jakoby and Pastan (eds), 1979, CellCulture, Methods in Enzymology, volume 58, Academic Press, Inc.,Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, HEK 293 cells, andWI38, BHK, and COS cell lines, although other cell lines may be used,such as cells designed to provide higher expression, desirableglycosylation patterns, or other features.

A number of viral vectors have been constructed, that can be used toexpress the disclosed polypeptides, including polyoma, i.e., SV40(Madzak et al., 1992, J. Gen. Virol., 73:1533-1536); adenovirus(Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner etal., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol.,66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA,89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson etal., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al.,1990, Hum. Gene Ther., 1:241-256); non-replicating adenoviruses ofchimpanzee origin (ChAdv; Tatsis et al., Gene Ther. 13:421-429, 2006);vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499);modified vaccinia Ankara (MVA) virus (Kremer et al., Methods Mol. Biol.890:59-92, 2012); adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282);herpes viruses, including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199); Sindbis viruses (Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879); alphaviruses (Schlesinger, 1993, Trends Biotechnol.11:18-22; Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377); and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al.,1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

III. Therapeutic Methods and Pharmaceutical Compositions

The immunogenic polypeptides disclosed herein (such as SEQ ID NOs: 1-8,or polypeptides having at least 95% sequence identity to SEQ ID NOs:1-8), or nucleic acids encoding the disclosed immunogenic polypeptides,can be administered to a subject to elicit an immune response in thesubject, such as an immune response to HIV. In some embodiments, one ormore of the disclosed polypeptides (or one or more nucleic acidsencoding the disclosed polypeptides) is administered to a subject withHIV infection or at risk of HIV infection. In other embodiments, the oneor more immunogenic polypeptides are administered to a subject as partof an immunization regimen. The one or more immunogenic polypeptides areadministered in an amount sufficient to elicit an immune response to HIVin the subject. In some examples, administration of the immunogenicpeptide inhibits (or in some instances even prevents) infection with HIVand/or reduces the signs and symptoms of HIV in an infected subject.

In particular embodiments, two or more of the disclosed polypeptides ornucleic acids encoding the polypeptides are administered to the subject.In some examples, the methods include administering to the subject oneor more Env mosaic proteins (or nucleic acids encoding at least twopolypeptides), for example, as a polyvalent immunogenic composition. Inparticular examples, the methods include administering to the subjectone or more of the Env mosaic proteins (for example, 2, 3, 4, 5, 6, 7,or more Env mosaic proteins) disclosed herein.

In some embodiments, a subject is administered a set of immunogenicpolypeptides, such as a set of three immunogenic polypeptides disclosedherein. In one example, a set of immunogenic polypeptides administeredto the subject includes three polypeptides comprising, consistingessentially of, or consisting of the amino acid sequences set forth asSEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In another example, a setof immunogenic polypeptides administered to the subject includes threepolypeptides comprising, consisting essentially of, or consisting of theamino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 4, and SEQ IDNO: 5. In a further example, a set of immunogenic polypeptidesadministered to the subject includes three polypeptides comprising,consisting essentially of, or consisting of the amino acid sequences setforth as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In additionalexamples, the C clade mosaic proteins disclosed herein could be usedsingly as SEQ ID NO: 1; as a pair of SEQ ID NO: 1 and SEQ ID NO: 2; oras a combination of all three proteins (SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO: 3) in a polyvalent vaccine. In further examples, SEQ ID NO: 4or SEQ ID NO. 5 could be utilized singly or as a pair (for example ingeographic regions where the B clade or CRF01 dominate the regionalepidemic), as well as the three protein combination of SEQ ID NO: 1, SEQID NO: 4, and SEQ ID NO: 5. One of skill in the art can identifyadditional combinations of the disclosed polypeptides that could beadministered to a subject as a set. In addition, the disclosed mosaicEnv proteins can be combined with natural strains to form additionalsets.

In some examples, the two or more immunogenic polypeptides (such as aset of immunogenic polypeptides) are administered simultaneously (forexample, as a mixture), substantially simultaneously (for example,within a few minutes of one another, such as within less than 5 minutesof one another), or sequentially (for example, within 5 minutes, 10minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, 24hours, or more of one another).

One or more of the disclosed polypeptides or nucleic acids encoding thepolypeptides (including vectors including the nucleic acid) can beadministered by any means known to one of skill in the art (see Banga,“Parenteral Controlled Delivery of Therapeutic Peptides and Proteins,”in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,Lancaster, Pa., 1995) either locally or systemically, such as byintramuscular, subcutaneous, or intravenous injection, but even oral,nasal, or anal administration is contemplated. In one embodiment,administration is by subcutaneous or intramuscular injection. To extendthe time during which the disclosed polypeptides are available tostimulate a response, the polypeptide or nucleic acid encoding thepolypeptide can be provided as an implant, an oily injection, or as aparticulate system. The particulate system can be a microparticle, amicrocapsule, a microsphere, a nanocapsule, or similar particle. (see,e.g., Banga, supra). A particulate carrier based on a synthetic polymerhas been shown to act as an adjuvant to enhance the immune response, inaddition to providing a controlled release. Aluminum salts can also beused as adjuvants to produce an immune response.

Optionally, one or more cytokines, such as interleukin (IL)-2, IL-6,IL-12, IL-15, RANTES, granulocyte macrophage colony stimulating factor(GM-CSF), tumor necrosis factor (TNF)-α, interferon (IFN)-α or IFN-γ,one or more growth factors, such as GM-CSF or G-CSF, one or morecostimulatory molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, orother B7 related molecules; one or more molecules such as OX-40L or 41BBL, or combinations of these molecules, can be used as biologicaladjuvants (see, for example, Salgaller et al., 1998, J. Surg. Oncol.68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1):S61-6;Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000,Adv. Exp. Med. Biol. 465:381-90) with the disclosed immunogenicpolypeptides. These molecules can be administered systemically (orlocally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B.7-2, OX-40L, 41 BBL,and/or ICAM-1 are administered.

Pharmaceutical compositions including the disclosed polypeptides, and/ornucleic acids encoding the polypeptides are also disclosed herein. Thepharmaceutical compositions can include one or more of pharmaceuticallyacceptable carriers, adjuvants (such as those described above), astabilizing detergent (such as polysorbate 80 (TWEEN® 80)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl); manufactured byICI Americas, Wilmington, Del.), TWEEN® 40, TWEEN® 20, TWEEN® 60,ZWITTERGENT® 3-12, TEEPOL® HB7, and SPAN® 85 detergents, for example, inan amount of approximately 0.05 to 0.5%, such as at about 0.2%), amicelle-forming agent (such as PLURONIC® L62LF, L101, and L64 blockcopolymer, polyethylene glycol 1000, and TETRONIC® 1501, 150R1, 701,901, 1301, and 130R1 block copolymer, for example, between 0.5 and 10%,or in an amount between 1.25 and 5%), and an oil (squalene, squalane,eicosane, tetratetracontane, glycerol, and peanut oil or other vegetableoils, for example, in an amount between 1 and 10%, or between 2.5 and5%). In one embodiment, the pharmaceutical composition includes amixture of stabilizing detergents, micelle-forming agent, and oilavailable under the name PROVAX® (IDEC Pharmaceuticals, San Diego,Calif.).

In some embodiments, a pharmaceutical composition includes one or morenucleic acids encoding a disclosed polypeptide. A therapeuticallyeffective amount of the nucleic acid(s) can be administered to a subjectin order to generate an immune response. In various embodiments, anucleic acid encoding a biological adjuvant (such as those describedabove) can be cloned into same vector as a nucleic acid encoding adisclosed polypeptide, or the nucleic acid can be cloned into one ormore separate vectors for co-administration. In addition, nonspecificimmunomodulating factors such as Bacillus Calmette-Guerin (BCG) andlevamisole can be co-administered.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, a nucleotide sequence encoding a disclosed polypeptidecan be placed under the control of a promoter to increase expression ofthe molecule. Immunization by nucleic acid constructs is well known inthe art and taught, for example, in U.S. Pat. No. 5,643,578 (whichdescribes methods of immunizing vertebrates by introducing DNA encodinga desired antigen to elicit a cell-mediated or a humoral response), andU.S. Pat. No. 5,593,972 and U.S. Pat. No. 5,817,637 (which describeoperably linking a nucleic acid sequence encoding an antigen toregulatory sequences enabling expression). U.S. Pat. No. 5,880,103describes several methods of delivery of nucleic acids encodingimmunogenic peptides or other antigens to an organism. The methodsinclude liposomal delivery of the nucleic acids (or of the syntheticpeptides themselves), and immune-stimulating constructs, or ISCOMs,negatively charged cage-like structures of 30-40 nm in size formedspontaneously on mixing cholesterol and Quil A™ (saponin).

In another approach to using nucleic acids for immunization, a disclosedimmunogenic polypeptide can also be expressed by attenuated viral hostsor vectors or bacterial vectors. Recombinant vaccinia virus,adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirusor other viral vectors (such as those described above) can be used toexpress the peptide or protein. For example, vaccinia vectors andmethods useful in immunization protocols are described in U.S. Pat. No.4,722,848. BCG (Bacillus Calmette Guerin) provides another vector forexpression of the peptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed immunogenicpolypeptide is introduced directly into cells. For example, the nucleicacid can be loaded onto gold microspheres by standard methods andintroduced into the skin by a device such as Bio-Rad's HELIOS™ Gene Gun.The nucleic acids can be “naked,” consisting of plasmids under controlof a strong promoter. Typically, the DNA is injected into muscle,although it can also be injected directly into other sites

The amount of the disclosed immunogenic polypeptide, or nucleic acidmolecule encoding the immunogenic polypeptide can vary depending uponthe specific polypeptide(s), the route and protocol of administration,and the target population. In some embodiments, each dose includes about1 μg to 1 mg of protein, such as from about 1 μg to about 500 μg, forexample, from about 1 μg to about 100 μg, or about 1 μg to about 50 μg,such as about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg,about 20 μg, about 25 μg, about 30 μg, about 40 μg, about 50 μg, about75 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, or about500 μg. An optimal amount for a particular composition can beascertained by standard studies involving observation of antibody titersand other responses in subjects (such as CTL or helper T cellresponses).

The disclosed Env mosaic proteins (such as a set of three mosaic Envpolypeptides) and/or nucleic acids encoding these proteins can be usedin a multistep immunization regime. In some examples, the regimeincludes administering to a subject a therapeutically effective amountof a first immunogenic polypeptide (or mixture or set of immunogenicpolypeptides) and boosting the immunogenic response with a secondimmunogenic polypeptide (or mixture or set of immunogenic polypeptides)after an appropriate period of time. This method of eliciting such animmune reaction is referred to as a “prime-boost” immunization regimen.Different dosages can be used in a series of sequential inoculations.Thus, a practitioner may administer a relatively large dose in a primaryinoculation (prime) and then boost with relatively smaller doses. Insome examples, the immunogenic polypeptide or mixture thereofadministered in both the prime and boost inoculations are the sameimmunogenic polypeptide or mixture thereof. In other examples, theimmunogenic polypeptide or mixture thereof administered in the boost isdifferent from that administered in the prime inoculation.

The prime can be administered as a single dose or multiple doses, forexample two doses, three doses, four doses, five doses, six doses, ormore can be administered to a subject over days, weeks or months. Theboost can be administered as a single dose or multiple doses, forexample two to six doses or more can be administered to a subject over aday, a week or months. Multiple boosts can also be given, such one tofive, or more. Different dosages can be used in a series of sequentialinoculations. For example a relatively large dose in a primaryinoculation and then a boost with relatively smaller doses. In someexamples, there are weeks (for example, at least one week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8weeks, at least 12 weeks, at least 16 weeks, at least 24 weeks, or more)between administration of a prime and a boost or between administrationof two boosts in a prime-boost regimen. The immune response against oneor more of the Env mosaic proteins can be generated by one or moreinoculations of a subject with an immunogenic composition disclosedherein.

The present disclosure is illustrated by the following non-limitingExamples.

EXAMPLES Example 1 B Cell-Optimized Mosaic Design Strategy

This example describes the design strategy used to develop Bcell-optimized mosaic Env proteins.

The B cell mosaic design was developed based on previous designstrategies utilized for T cell mosaic polypeptides (Fischer et al.,Nature Med. 13:100-106, 2007 and U.S. Pat. App. Publ. No. 2012/0231028,both of which are incorporated by reference herein in their entirety).The design strategy was modified as described below.

1. The capacity to incorporate structural information and alignments inthe genetic algorithm for mosaic design (FIG. 1). Structural informationwas used in several ways in the design strategy. Alignments as inputwere required to associate a position in the alignment with a positionon the structure. For every amino acid position in the alignment, the 10amino acid positions that were closest to it based on thethree-dimensional structure were defined, essentially defining aproximity sphere around every amino acid in the protein. The mosaicdesign code allows the user to define the dimensions of the proximitysphere by either choosing the number of the closest amino acids to beincluded, or by setting a spatially defined radius in angstroms; a 6.5angstrom radius and a 10 amino acid maximum was used in the firstdesigns. The positions in the proximity sphere were considered as a set,and all of the natural variation in each of those sets was determined,with the frequency of each form of the sphere calculated from thealignment. An additional constraint was added to the selection ofrecombinant mosaics—not only were breakpoints required to be spanned bysequences that are found in nature, but if a breakpoint occurred withina proximity sphere, it was also required that the new recombinant regionbe found in natural sequences. Co-optimization of the mosaic design togenerate polypeptide sets that provide optimal coverage of spatiallydefined antibody epitope-sized regions became the selection criterionfor the genetic algorithm. While most relevant B cell epitopes will beon the protein surface, including the constraint of finding common formsthat are represented in nature in the internal part of the protein,essentially requiring no local violation of natural forms in terms ofproximity spheres in any part of the structure, may help the mosaicconstructs retain structural integrity.

2. A strategy for spanning hypervariable loop regions for the creationof an intact vaccine. HIV has four hypervariable regions within thevariable loops V1, V2, V4, and V5 that typically evolve by insertion anddeletion rather than just by base substitution. These short regions varydramatically in length, charge, and number and location of N-linkedglycosylation site motifs. They impact HIV neutralizing antibodyrecognition, and are essentially not alignable so could not beincorporated into part (1) above. Thus all hypervariable regions wereexcised from the alignments for the mosaic design phase, and if aposition in the remaining core was close to these hypervariable regions,those positions were ignored during the mosaic design phase.

For the final designs, natural forms of these hypervariable regions thatwere short, relatively positively charged, with limited number ofpotential N-linked glycosylation sites (all desirable attributes interms of antibody recognition based on neutralization data from largepanels of antibodies compared to large panels of Envs tested inneutralization assays, provided by Dr. David Montefiori) werereintroduced. In addition, the frequencies of all peptide motifs of alllengths that were found within these regions were characterized, andnatural variants with common motifs to span the hypervariable loops werefavored.

3. A structural framework to define nearby regions in the protein.Ideally the HIV Env in its native form is needed, which requires atrimer structure. An early trimer model was used to enable the B cellmosaic design. The Env glycoprotein derived from a primary,neutralization-resistant (Tier 2) clade B isolate, HIV-1_(JR-FL), waschosen for structural analysis. The gp120-gp41 proteolytic cleavage sitein the HIV-1_(JR-FL) Env was eliminated by two single-residue changes(R508S and R511S in standard HXB2 numbering). To improve the expressionlevel on the cell surface, the gp41 cytoplasmic tail was truncatedstarting from Tyr 712. The modified Env, designated Env(−)ΔCT, thuscontained the complete ectodomain and transmembrane regions, and waspurified from the plasma membrane of Env-expressing cells aftersolubilization in Cymal-5 detergent. This procedure ensured that thepurified membrane-anchored Env(−)ΔCT trimers were glycosylated andpassed the quality-control checkpoints of the secretory pathway (Moulard& Decroly (2000) Biochem. Biophys. Acta 1469:121-132; Wyatt and Sodroski(1998) Science 280:1884-1888)) Importantly, HIV-1 Env(−)ΔCT complexespurified in this manner retain epitopes that are dependent uponconformation, glycosylation and quaternary structure. The detergentCYMAL®-5 was exchanged to CYMAL®-6 before preparation for cryo-EMimaging. The Env(-)ACT membrane glycoprotein, under the protection ofCYMAL®-6, was flash-frozen on holey carbon film-coated EM grids andcryo-EM image data were collected at liquid nitrogen temperature. Theimaging quality was found to be critically affected by the choice of thedetergent and its concentration in the vitrified cryo-EM samples. Adataset of 90,306 single-particle images was assembled and subjected tomultivariate data analysis, maximum-likelihood alignment andclassification (Frank, J Three-dimensional electron microscopy ofmacromolecular assemblies: visualization of biological molecules intheir native state. (Oxford Univ. Press, 2006); Sigworth (1998) J.Struct. Biol. 122:328-229; Scheres, et al. (2005) J. Mol. Biol.348:139-149).

An initial model was generated by angular reconstitution fromtwo-dimensional class averages refined by a maximum-likelihood approach(Sigworth et al. (1998) J. Struct. Biol. 122:328-339; Scheres et al.(2005) J. Mol. Biol. 348:139-149). The model was then further refined bya projection-matching algorithm to a final resolution of 10.8 Å,measured by Fourier shell correlation (FSC) with a 0.5-cutoff criterion(Liao & Frank (2010) Structure 18:768-775). Using the 10.8-Angstrommodel as a reference, by analyzing a large dataset of 582,914 individualEnv trimer images (equivalent to 1,748,742 protomers), a cryo-EM map wasobtained that was estimated to have a resolution of ˜6-Å based on the0.5-cutoff of Fourier shell correlation. A reference model, obtained byfiltering the ˜6-Å reconstruction to 8-Å, was used to align a largerdataset of about 1-million single-particle images by projectionmatching. Tens of iterations of angular refinement yielded areconstruction with an estimated resolution of ˜4 Å by Fourier ShellCorrelation 0.5-cutoff. The density map allowed an initial Cα model tobe traced manually in the program O (Jones, T. A. (2004) ActaCrystallogr. D 60:211-2125) (FIG. 2). Interpretation of the Cα model wasinitially assisted by comparisons with crystal structures of theCD4-bound HIV-1 gp120 core, primary sequence information, secondarystructure predictions by I-TASSER (Roy et al. (2010) Nat. Protoc.5:725-738) and PHYRE (Kelley & Sternberg (2009) Nat. Protoc. 4:363-371),and known patterns of Env variation, glycosylation and disulfide bondformation.

Example 2 Env Mosaic Proteins

This example describes the design of Env mosaic proteins.

Using the strategies described in Example 1, structure-based Env mosaicproteins were designed. The design was first optimized based onrestricting the design to a single HIV clade, a regional approach forSouthern African vaccine (C clade). A multi-clade global vaccine designwas also developed, one that used only Transmitted/Founder virussequences and one that used the full database. The Env structural mosaicprotein sequences for use in the initial testing phase are disclosedherein as SEQ ID NOs: 1-8. These proteins were stably expressed andcould be bound by critical broadly neutralizing antibodies, and sCD4. Inparticular, they were designed for use as sets of three proteins, andfor many of the antibodies tested, they showed differential affinity forthe antibody (see Example 3), a trait considered desirable andconsistent with the design strategy, in that each mosaic displaysdifferent but common epitope variants. The design was based on thehypothesis that exposure to the common epitope variants in a vaccinewill elicit antibodies with greater breadth, and the proteins disclosedherein were contemplated to be used in combinations of three proteins.Theoretical analysis suggests that the main advantage of the B cellmosaic design would be the possibility of a global vaccine, rather thana within-clade mosaic improving single-clade regional vaccine over a setof three natural C clade Envs (FIG. 3). The other B cell mosaicadvantage appears to be that they minimize the inclusion of rare andunique amino acids that could lead to type-specific response to avaccine.

A total of eight proteins were designed, which can be used incombinations of three antigens for use in single polyvalent vaccine.Particular combinations are described below.

1. Within clade C: Cmos3.1 (SEQ ID NO: 1), Cmos3.2 (SEQ ID NO: 2),Cmos3.3 (SEQ ID NO: 3). This set is a mosaic trio that was optimized tomaximize the coverage of C clade transmitted viruses from the CHAVI/CAVDSGA sequence collections. They were made serially, first optimizing thecoverage of Cmos3.1; then fixing it, and optimizing for the addition ofCmos3.2; then fixing both 3.1 and 3.2 and adding Cmos3.3. The serialaddition did not compromise the total coverage relative to optimizingthe three at once, and has the advantage of having the potential to beused as a single, double or tri-valent vaccine.

2. Three clade trimer: Cmos3.1 (SEQ ID NO: 1)+Bmos3.1 (SEQ ID NO:4)+CRF01mos3.1 (SEQ ID NO: 5). This combination uses the optimal singlemosaic from the transmitted virus sequences from three clades. Thecoverage of each of these clades is very good, and they were designedfrom transmitted viruses, which may be advantageous. The coverage ofother clades (A, G, F, CRF02, and others) was not optimal, but betterthan inter-clade coverage of with natural C clade antigens or C clademosaics (FIG. 3). If transmitted virus input proves to be important,this may be a good way to get expanded coverage based on the availabledata.

3. Global mosaics: Mmos3.1 (SEQ ID NO: 6), Mmos3.2 (SEQ ID NO: 7),Mmos3.3 (SEQ ID NO: 8). There were not enough acute transmitted/foundersequences to give broad weight to some important epidemic lineages:clades A, G, F, and CRF01 and CRF02 are under-represented in thetransmitted virus database, while B and C had a large enough sample sizeto work with. Thus chronic non-SGA sequences from the database were usedto supplement the input data set for the under-represented lineages tocreate a global design. Thus this mosaic design set gives expandedcoverage of all clades, but was not based on transmitted SGA sequences.

Example 3 Antigenicity Data

This example describes antigenicity of the global mosaic proteinsdescribed in Example 2.

The Mmos3.1, Mmos3.2, and Mmos3.3 polypeptides were expressed and testedin vitro using surface plasmon resonance (SPR). The dissociationconstant of the polypeptides for various antibodies and epitopes wasmeasured by SPR and is shown in Table 2. Low numbers representantibodies with a slower off rates for the proteins. The antibodiesnames are listed on the left, the region of the HIV Env the targetfollows in parenthesis. CD4 is the receptor molecule on the HIV targetedT cell in natural infection. 17b is a monoclonal antibody that binds tothe same region on the Env protein as the HIV-1 co-receptor molecule,and the capacity to bind to 17b and the co-receptor is CD4 inducible,hence the 17b (CD4i) nomenclature. The binding of CD4, and 17b after CD4is bound, indicates that these proteins are well folded, and have nativeconformation in these regions, and the Env proteins undergo abiologically appropriate conformational change when CD4 is bound. Theobservation that these antibodies bind well to these proteinsdemonstrates that despite being artificial mosaic constructs, they formcorrectly folded proteins and retain the three-dimensional antigenicdomains required for antibody binding.

TABLE 2 Antigenicity of B cell mosaic Envelopes using surface plasmonresonance Dissociation Constant Kd (nM) Mmos3.1 Mmos3.2 Mmos3.3Antibody/Epitope gp120 gp120 gp120 CD4 14.0* 2.9*  8.4* 17b (CD4i) +++**+++** +++** A32 (C1) <1*  0.2* <1*  VRC01 (CD4 bs) 5.7 19.1 3.4 19b (V3)3.3 32.7 8.4 PG9 (V1-V2) 339   8.5 369   CH01 (V1-V2) NB NB NB 697D (V2)5.0 121 165   2G12 (—CHO) 6.0 1543 412   CH58 (V2) NB 59.8 14.4  PGT1284.6 5.3 3.7 *Kd from single-shot kinetics **Relative to Con S gp140 NB =not bound

Each of the three polypeptides was titrated on PG9, a very potentHIV-specific neutralizing antibody that typically has high dissociationconstants (FIGS. 4A-C). Mmos3.2 bound extremely well and the epitopeexposure could trigger the B cell lineage. Mmos3.1 and Mmos3.3 arevariants that are common in nature, but they did not bind PG9 as well asMmos3.2. However, without being bound by theory, the presence of thesevariants in an immunogenic cocktail during affinity maturation couldenable antibodies to evolve that bind well to each variant, toleratingthe diversity (mimicking CH505). Mmos3.2 had a slow off rate for bindingto PG9. RV144 A244 was one of the few gp120s that bind well to PG9 (40nM). Mmos3.2 had a Kd of 8.5 nM (Table 1 and FIG. 4B), suggesting thatthe essential aspects of the epitope are present.

Binding of Mmos3.1, Mmos3.2, and Mmos3.3 to A32, sCD4, and T8 was tested(FIGS. 5-7). Each of the polypeptides was also titrated on HIV specificantibodies, including VRC01 (FIG. 8), 19 b (FIG. 9), CH01 (FILG. 10),697D (FIG. 11), 2G12 (FIG. 12), CH58 (FIG. 13), and PGT128 (FIGL. 14).

Example 4 Immunization of Animals

This example describes exemplary procedures for immunization of animalswith the disclosed immunogenic polypeptides. Although particular methodsare provided, one of ordinary skill in the art will appreciate thatadditional methods or variations of the described methods can also beutilized.

In some examples nucleic acid molecules encoding the disclosedimmunogenic polypeptides are cloned into a plasmid or a viral vector(such as an adenoviral vector or a modified Ankara vaccinia virusvector). Study animals (for example, mice or monkeys) are administeredplasmid or viral vector nucleic acid intramuscularly. Varying amounts ofthe nucleic acid can be administered, for example to test for anoptimally effective amount.

In other examples nucleic acid molecules encoding the disclosedimmunogenic polypeptides are cloned into an expression vector andexpressed in a host cell. The polypeptides are purified using standardmethods. Study animals (for example, mice or monkeys) are administeredthe polypeptides intramuscularly or subcutaneously. Varying doses areadministered to determine optimal amounts for eliciting an immuneresponse.

Immune responses elicited by the administered immunogenic polypeptidesare assessed. For example, cellular immune responses are assessed usingcytokine assays and/or interferon-γ ELISPOT assays. Humoral immuneresponses are assessed by direct ELISA utilizing one or more HIVproteins (such as a set of natural Env variants and/or the mosaic Envprotein(s) administered to the animal). Neutralization assays (forexample, a luciferase based pseudovirus neutralization assay) are alsoused to assess humoral immune responses.

Example 5 Treatment of HIV in a Subject

This example describes exemplary methods for treating or inhibiting anHIV infection in a subject, such as a human subject, by administrationof one or more of the immunogenic polypeptides or one or more nucleicacids encoding the immunogenic polypeptides disclosed herein. Althoughparticular methods, dosages and modes of administrations are provided,one skilled in the art will appreciate that variations can be madewithout substantially affecting the treatment.

Briefly, the method includes screening subjects to determine if theyhave HIV, such as HIV-1 or HIV-2. Subjects having HIV are selected forfurther treatment. In one example, subjects are selected who haveincreased levels of HIV antibodies in their blood, as detected with anenzyme-linked immunosorbent assay, Western blot, immunofluorescenceassay or nucleic acid testing, including viral RNA or proviral DNAamplification methods. In one example, half of the subjects follow theestablished protocol for treatment of HIV (such as a highly activeantiretroviral therapy). The other half follow the established protocolfor treatment of HIV (such as treatment with highly activeantiretroviral compounds) in combination with administration of theagents including a therapeutically effective amount of a disclosedimmunogenic polypeptide that induces an immune response to HIV. However,pre-screening is not required prior to administration of thecompositions disclosed herein.

In particular examples, the subject is treated prior to diagnosis ofAIDS with the administration of a therapeutically effective amount ofone or more of the disclosed immunogenic polypeptides. In some examples,the subject is treated with an established protocol for treatment ofAIDS (such as a highly active antiretroviral therapy) prior to treatmentwith the administration of a therapeutic agent that includes one or moreof the disclosed immunogenic polypeptides. However, such pre-treatmentis not always required and can be determined by a skilled clinician.

Following selection, an effective amount of one or more (such as three)immunogenic polypeptides disclosed herein, or one or more (such asthree) nucleic acids encoding disclosed immunogenic polypeptides isadministered to the subject (such as an adult human or a newborn infanteither at risk for contracting HIV or known to be infected with HIV).Additional agents, such as anti-viral agents, can also be administeredto the subject simultaneously or prior to or following administration ofthe disclosed agents. Administration can be achieved by any method knownin the art, such as oral, inhalation, intravenous, intramuscular,intraperitoneal, or subcutaneous administration.

The amount of the immunogenic polypeptides (or nucleic acids encodingthe polypeptides) administered to prevent, reduce, inhibit, and/or treatHIV or a condition associated with it depends on the subject beingtreated, the severity of the disorder and the manner of administrationof the immunogenic composition. Ideally, an effective amount of theimmunogenic composition is an amount sufficient to prevent, reduce,and/or inhibit, and/or treat the condition (for example, HIV) in asubject without causing a substantial cytotoxic effect in the subject.An effective amount can be readily determined by one skilled in the art,for example using routine trials establishing dose response curves. Inaddition, particular exemplary dosages are provided above. Thetherapeutic compositions can be administered in a single dose delivery,via continuous delivery over an extended time period, in a repeatedadministration protocol (for example, by a daily, weekly or monthlyrepeated administration protocol). In one example, a therapeuticallyeffective amount of a disclosed antigen that induces an immune responseto HIV is administered intravenously or intramuscularly to a human. Assuch, these compositions may be formulated with an inert diluent or witha pharmaceutically acceptable carrier. Immunogenic compositions can betaken long term (for example over a period of months or years).

Following the administration of one or more therapies, subjects havingHIV (for example, HIV-1 or HIV-2) can be monitored for reductions in HIVlevels, increases in a subjects CD4+ T cell count or reductions in oneor more clinical symptoms associated with HIV infection. In particularexamples, subjects are analyzed one or more times, starting 7 daysfollowing treatment. Subjects can be monitored using any method known inthe art. For example, biological samples from the subject, includingblood, can be obtained and alterations in HIV or CD4+ T cell levelsevaluated.

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same or a different schedule and/or preparationof agents that they previously received for the desired amount of time,including the duration of a subject's lifetime. A partial response is areduction, such as at least a 10%, at least 20%, at least 30%, at least40%, at least 50% or at least 70% reduction of HIV viral load, HIVreplication or combination thereof. A partial response may also be anincrease in CD4+ T cell count such as at least 350 T cells permicroliter.

Example 6 Treatment of Subjects

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from HIV that can be treated byeliciting an immune response, such as a neutralizing antibody responseto HIV.

In particular examples, the method includes screening a subject having,thought to have or at risk of having a HIV infection. Subjects of anunknown infection status can be examined to determine if they have aninfection, for example using serological tests, physical examination,enzyme-linked immunosorbent assay (ELISA), radiological screening, orother diagnostic techniques known to those of skill in the art. In someexamples, subjects are screened to identify a HIV infection, with aserological test, or with a nucleic acid probe specific for a HIVnucleic acid. Subjects found to (or known to) have a HIV infection canbe administered one or more disclosed immunogenic polypeptides (ornucleic acids encoding the polypeptides). Subjects may also be selectedwho are at risk of developing HIV for example, subjects exposed to HIV.

Subjects selected for treatment can be administered an effective amountof the disclosed immunogenic polypeptides or nucleic acids encoding thedisclosed immunogenic polypeptides. The particular dose can bedetermined by a skilled clinician. The polypeptides (or nucleic acids)can be administered in one or several doses, for example continuously,daily, weekly, or monthly. When administered sequentially the timeseparating the administration of the doses of the immunogenicpolypeptides can be seconds, minutes, hours, days, or even weeks.

Subjects are periodically tested for presence of HIV or HIV antibodiesin their blood, as detected with an enzyme-linked immunosorbent assay,Western blot, immunofluorescence assay or nucleic acid testing,including viral RNA or proviral DNA amplification methods.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

We claim:
 1. A set of isolated immunogenic polypeptides comprising: polypeptides comprising the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; polypeptides comprising the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 5; or polypeptides comprising the amino acid sequences set forth as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 2. An isolated immunogenic polypeptide comprising the amino acid sequence set forth as any one of SEQ ID NOs: 1-8 or an amino acid sequence having at least 95% identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-8.
 3. The isolated immunogenic polypeptide of claim 2, wherein the polypeptide consists of the amino acid sequence set forth as any one of SEQ ID NOs: 1-8.
 4. A set of isolated immunogenic polypeptides comprising two or more of the polypeptides of claim
 2. 5. The set of isolated immunogenic polypeptides of claim 4, wherein the two or more polypeptides are selected from polypeptides consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 1-8.
 6. A pharmaceutical composition comprising: one or more of the isolated immunogenic polypeptides of claim 2; and a pharmaceutically acceptable carrier.
 7. The pharmaceutical composition of claim 6, further comprising one or more of an adjuvant, a detergent, a micelle-forming agent, and an oil.
 8. A pharmaceutical composition comprising: the set of immunogenic polypeptides of claim 4; and a pharmaceutically acceptable carrier.
 9. A method for eliciting an immune response to human immunodeficiency virus (HIV) in a subject, comprising administering to the subject an effective amount of: the set of immunogenic polypeptides of claim 4; thereby eliciting an immune response to HIV in the subject.
 10. The method of claim 9, wherein the polypeptides in the set are administered to the subject simultaneously, substantially simultaneously, or sequentially.
 11. The method of claim 9, further comprising administering to the subject a therapeutically effective amount of an anti-viral agent.
 12. A method for eliciting an immune response to human immunodeficiency virus (HIV) in a subject, comprising administering to the subject an effective amount of the set of immunogenic polypeptides of claim 1, thereby eliciting an immune response to HIV in the subject.
 13. A method for eliciting an immune response to human immunodeficiency virus (HIV) in a subject, comprising administering to the subject an effective amount of the immunogenic polypeptide of claim 2, thereby eliciting an immune response to HIV in the subject. 